Pharmaceutical preparation containing plasminogen activators

ABSTRACT

The invention relates to pharmaceutical preparations containing plasminogen activators, sugars and tranexamic acid, in the form of a lyophilisate or an injection or infusion solution. In particular, the preparations contain a sugar, phosphate buffer, tranexamic acid as well as a surfactant and the liquid solutions preferably have a pH value of 5.5-6.5.

The invention relates to pharmaceutical preparations containingplasminogen activators or derivatives of same as the active substancesand to related pharmaceutical forms of administration in the form oflyophilisates or injection and infusion solutions, respectively.

The human tissue plasminogen activator (t-PA) is of immense therapeuticimportance in dissolving blood clots, e.g., in cardiac infarctions. t-PAcauses dissolving of blood clots by activation of plasminogen toplasmin. Plasmin, in turn, dissolves fibrin which is the major componentof the protein matrix of clotted blood.

Natural t-PA is composed of multiple functional domains F, E, K1, K2,and P. Domain P involves the proteolytically active centre causingcleavage of plasminogen to plasmin. The preparation of plasminogenactivators (PA) by genetic engineering, in particular, of t-PA orvarious t-PA muteins in eukaryotic and prokaryotic cells is alreadyknown. Here, in contrast to natural t-PA, t-PA derivatives aresynthesised from prokaryotes in non-glycosylised form.

In the meaning of the invention, in principle, such derivatives of t-PA,particularly those prepared by recombination, are possible as theplasminogen activators, which essentially comprise those protein regionsof natural protein that are responsible for the fibrinolysis of thethrombi. Here, such t-PA derivatives may also be used which havedeletions or substitutions of single or multiple amino acids in the t-PAsequence.

According to the invention, the following plasminogen activators may beemployed, for example: t-PA (e.g., Alteplase), LY 210825 (K2P availablefrom "Syrian hamster" cell lines, Circ. 1990, 82, 930-940); ΔFE3x andΔFE1x (K1K2P, Blood 1988, 71, 216-219); ΔFEK1 (K2P from C127 mousecells, J. Cardiovasc. Pharmacol. 1990, 16, 197-209); E-6010 (Jap. Circ.J. 1989, 53, p. 918); t-PA variants (Thromb. Haemost. 1989, 62, p. 542);K2P and D-K2P (Thromb. Haemost. 1989, 62, p. 393); MB-1018 (FK2K2P,Thromb. Haemost. 1989, 62, p. 543); FK2P (FASEB J. 1989, 3, A1031,abstract 4791); Δ1x (Circulation 1988, 4, II-15); K1K2P (Thromb. Res.1988, 50, 33-41); FK1K2P (J. Biol. Chem. 1988, 263, 1599-1602); TNKvariant of t-PA (WO 93/24635); bat-PA (Witt et al., Blood 1992, 79,1213-1217, and Mullot et al., Arterioscler. Thrombos. 1992, 12,212-221). In particular, those plasminogen activators are possible whichcontain the cringle 2 domain ("K2") of the t-PA and/or the serineprotease domain ("P"). In this respect, one may exemplify the K2P typet-PA mutein "r-PA" described in EP 0 382 174 (WO 90/09437).

More specifically, the present invention relates to K2P, K1K2P, FK1K2P,and FK2K2P type plasminogen activators as described in the followingprintings: Protein Engineering 5(1), 93-100 (1992); DE-OS-39 23 339.1;Circ. 1990, 82, 930-940; J. Cardiovasc. Pharmacol. 1990, 16, 197-203;Blood 1988, 71, 216-219; J. Biol. Chem. 1988, 263, 1599-1602; Thromb.Haemost. 1989, 62, p. 543. In particular, recombinant K2P typeplasminogen activators are employed as described in EP-A-0 382 174 andin Protein Engineering Vol. 5(1), p. 93-100 (1992). Further t-PA muteinsof this type are described in the following patent applications: U.S.Pat. No. 4,970,159, EP-A-0 196 920, EP-A-0 207 589, AU 61804/86, EP-A-0231 624, EP-A-0 289 508, JP 63133988, EP-A-0 234 051, EP-A-0 263 172,EP-A-0 241 208, EP-A-0 292 009, EP-A-0 297 066, EP-A-0 302 456, EP-A-0379 890.

The substantial influence of the sugar proportion on solubility andaggregation of proteins is known from prior art (J. Biol. Chem. 263(1988), 8832-8837). Thus, it has been determined in EP-B-458 950 that,e.g., a non-glycosylated recombinant plasminogen activator of domaincomposition K2P has a substantially poorer solubility than glycosylatedt-PA derivatives, for example. As a rule, non-glycosylated plasminogenactivators such as r-PA dissolve only to a slight extent in buffersconventionally used for the solubilisation of proteins, such as 50mmoles/l of Na citrate pH 6, 50 mmoles/l of phosphate buffer orphysiological NaCl solution. However, utilisation as a therapeutic agentrequires plasminogen activators to be present at sufficiently highconcentrations, preferably, at a concentration of up to 10 mg/ml.

Increasing the solubility of t-PA from prokaryotes by neutral orslightly alkaline arginine formulations is known from EP-A-0 217 379.However, this procedure suffers from the drawback that good solubilityof t-PA from prokaryotes can only be achieved with very high arginineconcentrations.

Further galenic formulations of plasminogen activators or theirderivatives are known from WO 90/01333, WO 89/050347, WO 90/08557, EP 0297 294, EP 0 156 169, and EP 0 228 862.

Thus, WO 90/01333 (Invitron) describes the combination of lysine,histidine, arginine with citrate for t-PA and derivatives from bacteria.Citrate is used at 5 mmoles/l, lysine, histidine, arginine at 150mmoles/l at pH 6. Furthermore, albumin is added.

WO 89/050347 (Invitron) describes the combination of arginine (20-200mmoles/l) and citrate (20-80 mmoles/l) at pH 5-8.

WO 90/08557 (Genetics Institute) discloses a combination of creatininewith various additives such as histidine, arginine, proline, betaine,choline, imidazole, tryptophan, citrate, optionally with addition ofglutamic acid, aspartic acid and succinic acid.

EP 0 297 294 (Behring) discloses the combination of at least two aminoacids such as lysine, ornithine, arginine, tranexamic acid and otheradditives at pH 5-10.

EP 0 156 169 (Asahi) describes ornithine and/or lysine, optionally withaddition of citrate, glycine or phosphate, and EP 0 228 862 (Genentech)discloses a formulation containing arginine with or without chloride andwith or without detergent.

Likewise, alternative formulations of r-PA in the presence of lysine andlysine analogues, respectively, in solutions buffered with citric acidare described in EP 0 458 950 (Boehringer Mannheim). However, morerecent examinations demonstrate that the solubility of r-PA in theseformulations as well, is not as yet completely sufficient. It has beendetermined that in fact, the solubility may be improved by increasingthe citrate concentration; however, such formulations are not or only inpart tolerated by the veins. Moreover, the high salt concentrations andthe low glass temperature (Tg') associated therewith result in thenecessity to carry out the lyophilisation of these formulations attemperatures below -45° C. and -50° C. Technically, these temperaturesare realizable often only at a very high expenditure, and they requirecomplex control and monitoring elements in order to control and measurethe optimum conditions for lyophilisation. Moreover, this involves arelatively high expenditure of energy. Furthermore, for industrialproduction there are often used rather old lyophilisation plants, whichlack the requisite complex measuring and control technology, and where,consequently, as a rule, there can only be guaranteed a workingtemperature of about -45° C.

The object of the invention is to provide formulations of plasminogenactivators and their derivatives which are well tolerated by the veins,contain the active substance at sufficiently high concentrations andensure good solubility of the active substance, with the intention ofmaintaining stability of the PA in the lyophilisate over a prolongedperiod of time. In addition, the invention is aimed to developformulations which can be lyophilised reliably on an industrialproduction scale as well, with the intention of ensuring a wellreproducible product quality of the lyophilisates.

The object of the invention is attained by a pharmaceutical preparationcontaining a plasminogen activator, with the preparation containing atleast one sugar and tranexamic acid as pharmaceutical additives. Inlyophilised form, the preparation is stable on storage over a prolongedperiod of time. Likewise, the aqueous injection solution prepared byreconstitution meets this criterion of improved storage stability. Inparticular, this is the case when the pH value of the solution isadjusted to a value of between 5.5-6.5. As further additives, thepharmaceutical preparations may contain conventional buffer substancesor surface-active materials (anionic, cationic or neutral surfactants).

As an example of the plasminogen activators (PA) possible within themeaning of the invention, the plasminogen activator K2P (BM 06.022)described in more detail in the European patent application EP-A-0 382174 was used. It consists of the cringle 2 (K2) and the protease domain(P) of human t-PA and, due to its expression in Escherichia coli cells,is present in non-glycosylated form.

As sugar, the formulations of the invention preferably contain mono- ordisaccharides. As the disaccharides, particularly saccharose, trehalose,maltose or lactose are possible. In particular, the monosaccharides aregalactose or corresponding amino sugars such as, e.g., galactosamine.Preferably, the non-reducing sugars saccharose and trehalose areemployed. The sugar concentration in the aqueous injection solution ispreferably 40-100 mg/ml, with between 50-70 mg/ml being preferred.

As buffer substances, the pharmaceutical preparations may containconventional substances possible for such purposes and representingsalts of strong acids with weak bases or of weak acids with strongbases. As examples, there may be mentioned: alkali salts of phosphoricacid, tartaric acid, malic acid and the like. Amino acids are alsopossible. Preferably, the preparations according to the inventioncontain phosphate buffer. In particular, the concentration of phosphatebuffer in the aqueous solution ready for injection is 50-300 mmoles/l,preferably 80-220 mmoles/l and, particularly preferred, 130-170mmoles/l. As a phosphate buffer, disodium or dipotassium hydrogenphosphate is preferably used, which is adjusted to the respective pHvalue using phosphoric acid.

As solubiliser, the preparations according to the invention containtranexamic acid (TES). The concentration of tranexamic acid in theaqueous solution ready for injection is preferred to be 1-50 mmoles/l,preferably 8-12 mmoles/l. Particularly advantageous is the use of aconcentration of 10 mmoles/l. Surprisingly, tranexamic acid has beenfound to increase the solubility of plasminogen activators, particularlyof non-glycosylated plasminogen activators in an aqueous medium. Forinstance, in the case of the plasminogen activator derivative r-PA,solubility was increased at least by a factor of 1.5 with respect to asolution free of tranexamic acid. The factor for the solubility increasepreferably ranges from 2-3. This effect, above all, is advantageouswithin the scope of the preparation of lyophilised pharmaceuticalpreparations of plasminogen activators since, due to the possibility ofincreasing the concentration of the plasminogen activator in thesolutions used, the amount of water to be used in the production processmay be reduced significantly and, on the whole, a production is achievedwhich is energy-saving and more cost effective. In particular, thelyophilisation periods in the production process are reduced.

As surfactants, non-ionic, anionic or cationic surfactants, preferably,however, non-ionic surfactants such as Tween 80 or Tween 20 may be used.The surfactant concentration is 0.005-0.1% (w/v) and preferably 0.01%.

A pH value of between 5.5-6.5 is suitable for a pharmaceuticalpreparation in aqueous form according to the invention; a pH value of5.8-6.2 is preferred.

The preparations of the invention contain the active substance at aconcentration of up to 10 mg/ml, preferably of 3-5 mg/ml.

The pharmaceutical preparations according to the invention are employedas injection or infusion solutions. This can be done by providing asolution ready for injection, which has the composition according to theinvention. However, the pharmaceutical preparations may also be providedin the form of lyophilisates. These are then reconstituted using per seknown agents or solutions (e.g., with water) suitable for injections. Asthe injection medium, water is preferably employed for the preparationsof the invention, which optionally may contain conventional isotonicadditives such as a physiological concentration of NaCl.

Likewise, the invention is directed to the process of manufacturing apharmaceutical preparation containing plasminogen activators orderivatives of same having a pH value of from 5.5 to 6.5 and their usein manufacturing the pharmaceutical preparations according to theinvention.

In addition, the invention is directed to the use of a specified mixtureof pharmaceutical adjuvants consisting of the group sugar and tranexamicacid for the longterm stabilisation of plasminogen activators. Inparticular, in order to achieve good storage stability, a combination ofthe adjuvants sugar, phosphate buffer, tranexamic acid, and surfactantis especially advantageous.

Examinations on stability show that the preparations according to theinvention are stable as solutions for at least 30 days at 4° C. Thestability of the active substance in the lyophilisates according to theinvention is between two and five years at 4° C. Of particularlyadvantageous influence is the fact that the drug preparations haveexcellent stability even at increased thermal stress, which makes themlargely insensitive to temperature fluctuations which, in particular,may occur in warmer climatic regions of the earth, and all the more sowhen the cold chain is interrupted. Due to the often long shippingroutes involved in pharmaceutical forms of administration from the drugmanufacturer down to the consumer in the various countries, unintendedexposure of the preparations to temperature fluctuations, possibly evenover a prolonged period of time, cannot be excluded, which give rise toactivity loss of the protein and thus, in the extreme case, render thesuccess of therapeutic treatment questionable. However, thepharmaceutical forms of administration according to the invention arelargely insensitive to such temperature fluctuations. It has beenpossible to demonstrate in preliminary examinations on stability that inthe preparations, even when exposed to a temperature of 35° C. over atime period of 30 days, no significant impairment regarding thestability of the protein could be detected. Therefore, the preparationsmust be assumed to have a stability of from two to five years whenstored properly at refrigerator temperature.

Furthermore, the preparations according to the invention areadvantageous in that the glass temperature (Tg') of the frozen solutionfor the preparation of the lyophilisates is relatively high, rangingbetween -33° C. and -40° C. so that the reproducibility of productquality of the lyophilisates is ensured with sufficient reliability onthe production scale as well. The relatively high glass temperature isadvantageous especially because if technically induced or unforeseenincreases in temperature occur during the often very long lyophilisationperiods, the risk of unintended thawing of the frozen solutions is lessprobably involved. In cases where the glass temperature is below -40° C.and that temperature is exceeded during the lyophilisation process,which is particularly the case in technical lyophilisation plants, whichwork at temperatures somewhat close to -45° C., the undesired exceedingof the glass temperature may cause disadvantageous changes in the frozensolution. Sufficiently good product quality is then no longer ensured.Here loss of activity of the protein, and the formation of aggregates,respectively, may occur, in particular, in the case ofprotein-containing lyophilisates. A further advantage in the case of thepreparations according to the invention, with a glass temperature offrom -33° C. to -40° C., is that the requisite expenditure of energy forthe lyophilisation process is minimised because the lyophilisationprocedure need not necessarily be carried out at temperatures below -50°C.

In the meaning of the present invention, relatively high glasstemperatures can be attained for the frozen solutions especially when apotassium salt such as dipotassium hydrogen phosphate or potassiumdihydrogen phosphate is used as the phosphate buffer for the solutionsrequired for lyophilisation. In this regard, in particular, dipotassiumhydrogen phosphate at a concentration of from 20-40 mg/ml, preferably20-30 mg/ml can be used. The increase in glass temperature can beenhanced by adding saccharose. In this connection, saccharose is addedpreferably at a concentration of from 60-90 mg/ml, in particular, 60-80mg/ml. The use of a solution containing about 26 mg/ml of dipotassiumhydrogen phosphate (K₂ HPO₄ ×12 H₂ O) and about 70 mg/ml of saccharoseis particularly preferred. The pH value of the solution is adjusted to avalue of 6 using, preferably, 85%-phosphoric acid (about 12 mg/ml). Thesolution further contains about 0.5-5 mg/ml, preferably 1-2 mg/mltranexamic acid, in particular, about 1.6 mg/ml. Besides this, thesolution may contain, in addition, surfactants such as Tween 80, whichis applied preferably at a concentration of from 0.01-0.3 mg/ml, inparticular, about 0.1 mg/ml.

Another advantage of the lyophilisates according to the invention isthat on completion of the lyophilisation they exhibit a relatively lowresidual moisture. The residual moisture is, in particular, at valuesbelow 5%, preferably ranging between 0.5-4%, in particular, 1-3%. Thevalues for lyophilisates usually are at levels above 6%. The lowresidual moisture contributes to the improved storage stability of thepreparations. Preparations having a higher residual moisture oftenresult in instability of the protein, which becomes apparent from theloss of biological activity or the formation of aggregates.

Another advantage of the preparation according to the invention is thatwhen producing the lyophilisates, due to the increase in solubility ofnon-glycosylated plasminogen activators, in particular, in the case ofthe type K2P, K1K2P or P type muteins, achieved by the addition oftranexamic acid, one may start with smaller volumes (for example, ofabout 5 ml per single administration form) of solutions to be frozen, sothat the lyophilisation period is significantly reduced with respect tosolutions hitherto used for the preparation of lyophilisates (forexample, of about 10 ml per administration form). Preferably, one startswith solutions containing the active substance at a concentration abouttwice as high as compared to the aqueous administration form ready forinjection so that aqueous solutions of 5 ml can be used instead of thehitherto conventional solutions of 10 ml. "It is most preferred where,prior to lyophilization, the aqueous solution contains the activesubstance at a concentration which is up to three times higher than theconcentration of a ready-to-use aqueous injection or infusion solution."

Examinations on venous tolerance demonstrate very good tolerance of thepreparations according to the invention.

Without limiting, the following embodiments are intended to illustratethe invention in more detail.

EXAMPLE 1

Clouding on Mechanical Stress/Measurement of Light Scattering ofPreparations According to the Invention

r-PA (BM 06.022) was adjusted to a protein concentration (C_(prot).) of6 mg/ml (ultrafiltration over an Amicon YM 10 membrane) and dialysedagainst the buffers as indicated. Subsequently, the samples wereadjusted to C_(prot). =4 mg/ml and were a) left unchanged, b) filled upwith 0.01% Tween 80, and c) with 0.01% Tween 20.

Exposing each of the samples to stress was effected for 10 s on a WhirlMix (Janke & Kunkel, IKA Labortechnik VF2, maximum rotational speed).Subsequently, the samples were incubated for 2 minutes at roomtemperature.

Light scattering of the unstressed and stressed samples was measured byfluorimetry at 25° C. (Ex. 360 nm, Em.: 360 nm; Ex. bandpath: 3 nm; Em.bandpath: 10 nm; measuring intervals: 10 s over 3 min). In FIG. 1 thevalues for the scattering of the samples are given, each one correctedby the fluorescence of the buffer.

Result:

The present data shows that without addition of detergents, there issubstantial increase in light scattering of the sample under mechanicalstress (FIG. 1). By adding detergents, the increase can be suppressed tothe largest extent. Within the scope of measuring accuracy, nodifference between Tween 80 and Tween 20 is notable.

In FIG. 1 (mechanical stressing of samples A, B, C, D) the obtainedresults are summarised. The meanings of each of the abbreviations usedin FIG. 1 are as follows:

    ______________________________________                                        A)      150        mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                 10         mM of TES                                                          50         mg/ml of saccharose                                                           a) without detergent                                                          b) with 0.01% of Tween 80                                                     c) with 0.01% of Tween 20                                  B)      150        mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                 10         mM of TES                                                          50         mg/ml of trehalose                                                            a) without detergent                                                          b) with 0.01% of Tween 80                                                     c) with 0.01% of Tween 20                                  C)      150        mM of K.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                  10         mM of TES                                                          50         mg/ml of saccharose                                                           a) without detergent                                                          b) with 0.01% of Tween 80                                                     c) with 0.01% of Tween 20                                  D)      150        mM of K.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                  10         mM of TES                                                          50         mg/ml of trehalose                                                            a) without detergent                                                          b) with 0.01% of Tween 80                                                     c) with 0.01% of Tween 20                                  ______________________________________                                    

EXAMPLE 2

Storage of r-PA (BM 06.022) in TES/Saccharose-Containing FormulationsAccording to the Invention//Multiple Freezing and Thawing

r-PA was dialysed against the buffer (without Tween 80) indicated inTable 1, adjusted to C_(prot). =4 mg/ml, filled up with Tween 80 toC=0.01%, portioned, and sterile filtrated. Each time 9 samples werefrozen at -20° and -70° C., respectively. On the days as indicated inthe Table all of the samples were thawed (15 min at 25° C. in a waterbath) except for the control. Each time one sample was analysed(C_(prot). and amidolytic activity). The rest of the samples were frozenat -20° C. and -70° C., respectively. After completion of the series theactivity of the non-stressed sample was determined.

Result:

As it is apparent from the data in Table 1, r-PA may be frozen andthawed at least eight times without loss of activity.

                  TABLE 1                                                         ______________________________________                                                150 mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                        10 mM of TES                                                                  50 mg/ml of saccharose                                                        0.01% of Tween 80                                                     -20° C.      -70° C.                                                   C.sub.prot.                                                                             AA         C.sub.prot.                                                                           AA                                        Day     mg/ml!    MU/ml!     mg/ml!  MU/ml!                                   ______________________________________                                        0      4.1       3.1        4.1     3.1                                       1      4.5       3.1        4.6     3.0                                       2      4.6       3.2        4.8     3.1                                       3      4.3       3.1        4.0     3.0                                       4      4.8       3.1        4.7     3.1                                       5      4.1       3.3        4.1     3.2                                       6      4.2       3.0        4.2     3.1                                       7      4.1       3.1        4.3     3.2                                       8      4.0       2.9        4.3     3.1                                       Control                                                                              4.3       3.0        4.3     3.1                                       ______________________________________                                        Control:                                                                            Untreated sample measured                                               AA:   Amidolytic activity                                                     C.sub.prot. :                                                                       Protein concentration                                               

EXAMPLE 3

Stability of r-PA in Solution

Comparison of Various Formulations at C_(prot). =4 mg/ml

r-PA (BM 06.022) was concentrated over an Amicon YM 10 membrane to 5mg/ml and dialysed against the buffer (without Tween 80) indicated inTable 2. The dialysates were adjusted to C_(prot). =4 mg/ml, filled upwith Tween 80 to C=0.01%, portioned, and stored at -20° C. and 4° C.After 7, 14, 20 and 30 days, amidolytic activity and proteinconcentration of the stressed samples were determined.

Result:

The samples stored at -20° C. and 4° C. remain unchanged over 30 days.

                  TABLE 2                                                         ______________________________________                                                150 mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                        10 mM of TES                                                                  50 mg/ml of trehalose                                                         0.01% of Tween 80                                                            -20° C.   +4° C.                                                  C.sub.prot.                                                                           AA         C.sub.prot.                                                                         AA                                          Day       mg/ml!  MU/ml!     mg/ml!                                                                              MU/ml!                                     ______________________________________                                         0       3.9     2.1                                                           7       3.9     2.3        3.97  2.4                                         14       3.9     2.3        3.98  2.4                                         20       3.9     2.3        3.9   2.4                                         30       3.9     2.3        3.9   2.4                                         ______________________________________                                         AA: Amidolytic activity C.sub.prot. : Protein concentration              

EXAMPLE 4

Stability of r-PA in Solution

Comparison of Various Formulations at C_(prot). =6 mg/ml

r-PA (BM 06.022) was dialysed overnight against the buffers indicatedbelow and adjusted to C_(prot). =6 mg/ml by concentrating over an AmiconYM 10 membrane. The samples were filled into portions of 1 ml and storedfor 30 days at -20° C. and 4° C. After 1, 2, 5, 9, 15 and 29 daysamidolytic activity and protein level were determined each time.

                  TABLE 3                                                         ______________________________________                                                200 mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                        10 mM of TES                                                                  50 mg/ml of saccharose                                                        0.01% of Tween 80                                                            -20° C.                                                                             +4° C.                                                      C.sub.prot.                                                                           AA         C.sub.prot.                                                                         AA                                          Day       mg/ml!  MU/ml!     mg/ml!                                                                              MU/ml!                                     ______________________________________                                        0        6.0     3.3        6.0   3.3                                         1        5.7     3.3        5.7   3.6                                         2        5.8     3.3        5.9   3.4                                         5        5.9     3.2        6.0   3.6                                         9        6.0     3.3        5.9   3.7                                         15       5.8     3.5        5.9   3.5                                         29       5.9     3.6        5.9   3.8                                         ______________________________________                                         AA: Amidolytic activity C.sub.prot. : Protein concentration              

                  TABLE 4                                                         ______________________________________                                                150 mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                        10 mM of TES                                                                  50 mg/ml of saccharose                                                        0.01% of Tween 80                                                            -20° C.                                                                             -70° C.                                                     C.sub.prot.                                                                           AA         C.sub.prot.                                                                         AA                                          Day       mg/ml!  MU/ml!     mg/ml!                                                                              MU/ml!                                     ______________________________________                                        0        5.9     3.5        5.9   3.5                                         1        5.5     3.5        5.6   3.4                                         2        5.6     3.4        5.5   3.5                                         5        6.0     3.5        6.2   3.6                                         9        6.0     3.3        6.0   3.3                                         15       6.0     3.6        5.9   3.1                                         29       5.9     3.5        6.0   3.6                                         ______________________________________                                         AA: Amidolytic activity C.sub.prot. : Protein concentration              

Result:

The Tables indicate that the active substance BM 06.022 in theformulations mentioned is stable for at least 29 days at -20° C. and 4°C.

EXAMPLE 5

Solubility of r-PA in the Formulations of the Invention

r-PA (BM 06.022) was concentrated to 6 mg/ml over YM 10 and dialysedagainst the buffer indicated below. The dialysates in turn wereconcentrated over an Amicon YM 10 membrane until clouding occurred.Following centrifugation of the samples, the supernatants were storedfor five days at 4° C. Amidolytic activity and C_(prot). were determinedat the beginning and the end of storage.

                  TABLE 5                                                         ______________________________________                                                150 mM of Na.sub.2 HPO.sub.4 /H.sub.3 PO.sub.4, pH 6.0                        10 mM of TES                                                                  50 mg/ml of saccharose                                                        0.01% of Tween 80                                                                    C.sub.prot.                                                                           AA                                                     Day             mg/ml!  MU/ml!                                                ______________________________________                                        0              10.2    4.8                                                    5              10.2    4.9                                                    ______________________________________                                         AA: Amidolytic activity C.sub.prot. : Protein concentration              

Result:

The solubility of r-PA is about 10 mg/ml. At maximum proteinconcentration the sample may be stored at least for up to 5 days at 4°C. without change in amidolytic activity.

EXAMPLE 6

Preparation of Liquid Administration Forms

The following administration forms were prepared as solutions prior tolyophilisation:

Composition of the solutions prior to lyophilisation

    ______________________________________                                        Solution A:                                                                   BM 06.022      4            mg/ml                                             Na.sub.2 HPO.sub.4.12H.sub.2 O                                                               53.72        mg/ml                                             H.sub.3 PO.sub.4 85%                                                                         11.3         mg/ml                                             Tranexamic acid                                                                              1.6          mg/ml                                             Saccharose     50           mg/ml                                             Tween 80, pH 6 0.1          mg/ml                                             Solution B:                                                                   BM 06.022      4            mg/ml                                             K.sub.2 HPO.sub.4                                                                            26.2         mg/ml                                             H.sub.3 PO.sub.4 85%                                                                         11.6         mg/ml                                             Tranexamic acid                                                                              1.6          mg/ml                                             Saccharose     70           mg/ml                                             Tween 80, pH 6 0.1          mg/ml                                             ______________________________________                                    

Preparation of the Lyophilisates:

Following sterile filtration, aliquots of each 5 ml are filled into 20ml glass vials. The lyophilisation is conducted as follows: The filledglass vials are placed in a freeze drier and frozen for 10 hours attemperatures of from -40° to -50° C. plate temperature and atmosphericpressure. Subsequently, a vacuum value of p=0.01 to 1 mbar is applied tothe chamber. Thereafter, the plate temperature is adjusted so that atany time the product temperature will be reliably below the respectiveglass transition temperature. Once the total amount of ice has sublimedoff, the plate temperature is raised to a value of 20°-40° C. andsubsequently, an afterdrying is carried out in a vacuum. The residualmoisture was determined according to conventional methods (determinationaccording to the method of Karl Fischer) and is 6% for solution A, and3% for solution B. Storage of the lyophilisates for examinations onstability is conducted at temperatures of 4° C. (refrigeratortemperature), 20° C. (room temperature) and 35° C. Stability of thelyophilisates after a storage period of four to twelve weeks is met atthe indicated temperatures as it is apparent from the analyticalexaminations regarding amidolytic activity and protein concentration.

Reconstitution of the lyophilisates for application:

The lyophilisates are filled up to 10 ml with water for injections. Thiscorresponds to a dilution by a factor of two in relation to the originalvolume of the solution prior to lyophilisation.

EXAMPLE 7

Test on Venous Tolerance of the Formulations of the Invention

Two real solutions and two placebo solutions having the compositionslisted in Table 6 were prepared and administered intravenously torabbits. An application amount of 0.5 ml/animal was used; five animalswere used for each of the four test solutions.

                  TABLE 6                                                         ______________________________________                                        Composition of Real and Placebo Solutions                                     ______________________________________                                        1. Real Solutions                                                                          Test No. 93/1292                                                                            Test No. 93/1294                                   ______________________________________                                        BM 06.022    10       MU       10     MU                                      Dipotassium hydrogen                                                                       131.0    mg       131.0  mg                                      phosphate                                                                     Phosphoric acid 85%                                                                        58       mg       58     mg                                      Tranexamic acid                                                                            8.0      mg       8.0    mg                                      Saccharose   250.0    mg       350.0  mg                                      Polysorbate 80                                                                             0.5      mg       0.5    mg                                      Water p.i.   q.s. 10.0                                                                              ml       q.s. 10.0                                                                            ml                                      pH           6.0               6.0                                            Osmolarity   320      mOsmoles 351    mOsmoles                                ______________________________________                                        2. Placebo Solutions                                                                       Test No. 93/1291                                                                            Test No. 93/1293                                   ______________________________________                                        Dipotassium hydrogen                                                                       131.0    mg       131.0  mg                                      phosphate                                                                     Phosphoric acid 85%                                                                        58       mg       58     mg                                      Tranexamic acid                                                                            8.0      mg       8.0    mg                                      Saccharose   250.0    mg       350.0  mg                                      Polysorbate 80                                                                             0.5      mg       0.5    mg                                      Water p.i.   q.s. 10.0                                                                              ml       q.s. 10.0                                                                            ml                                      pH           6.0               6.0                                            Osmolarity   322      mOsmoles 343    mOsmoles                                ______________________________________                                    

Result:

The animals' reactions on injection and the histological findingsdemonstrate that all of the employed test solutions are well tolerated.

We claim:
 1. A pharmaceutical composition comprising a protein havingplasminogen activator activity, sugar and tranexamic acid, wherein saidtranexamic acid is the only aminocarboxylic acid in said composition,and wherein said composition is devoid of citrate.
 2. The compositionaccording to claim 1, wherein said sugar is a disaccharide.
 3. Thepreparation according to claim 2, wherein said disaccharide issaccharose or trehalose.
 4. The composition according to claim 1,further comprising a buffer, wherein said buffer comprises a potassiumsalt of phosphoric acid.
 5. The composition according to claim 4,wherein said potassium salt of phosphoric acid is dipotassium hydrogenphosphate.
 6. The composition according to claim 4, wherein said bufferis 50-300 mmoles/l of phosphate buffer.
 7. The composition according toclaim 6, wherein said buffer is 80-220 mmoles/l of phosphate buffer. 8.The composition according to claim 1, wherein said composition is in theform of a frozen solution.
 9. The composition according to claim 1,wherein said sugar is present in an amount of 40-100 mg/ml.
 10. Thecomposition according to claim 7, wherein said sugar is present in anamount of 50-70 mg/ml.
 11. The composition according to claim 1, whereinsaid tranexamic acid is present in an amount of 1-50 mmoles/l.
 12. Thecomposition according to claim 11, wherein said tranexamic acid ispresent in an amount of 8-12 mmoles/l.
 13. The composition according toclaim 1, further comprising 0.005-01% of a surfactant.
 14. Thecomposition according to claim 13, wherein said surfactant is present inan amount of 0.01%.
 15. The composition according to claim 1, whereinsaid protein having plasminogen activator activity is a K2P typerecombinant protein having plasminogen activator activity.
 16. Thecomposition according to claim 15, wherein said K2P type recombinantprotein having plasminogen activator activity is BM 06.022.
 17. Thecomposition according to claim 1, wherein said protein havingplasminogen activator activity is t-PA at a concentration of up to 10mg/ml.
 18. The composition according to claim 1, wherein saidcomposition is a lyophilizate.
 19. The composition according to claim 1,wherein said composition is an aqueous solution and the pH value of thesolution is 5.5-6.5.
 20. The composition according to claim 1, whereinsaid composition has a storage stability of 30 days at a temperature of35° C.
 21. A composition in the form of a lyophilisate with a residualmoisture content of less than 5%, comprising a protein havingplasminogen activator activity, sugar, phosphate buffer, tranexamic acidand a surfactant, wherein said composition is devoid of citrate andwherein said tranexamic acid is the only aminocarboxylic acid in saidcomposition.
 22. The composition according to claim 21, wherein saidlyophilisate has a residual moisture between 1-3%.
 23. The compositionaccording to claim 21, wherein said composition has a storage stabilityof 30 days at a temperature of 35° C.
 24. A process for preventing theloss of plasminogen activator activity or for preventing the formationof protein aggregates in a composition which contains a protein havingplasminogen activator activity, comprising mixing said protein havingplasminogen activator activity with sugar and tranexamic acid, whereinsaid tranexamic acid is the only aminocarboxylic acid in saidcomposition, and wherein said composition is devoid of citrate.
 25. Aprocess for preparing a lyophilized pharmaceutical compositioncomprising a protein having plasminogen activator activity, sugar andtranexamic acid, wherein said tranexamic acid is the onlyaminocarboxylic acid in said composition, and wherein said compositionis devoid of citrate, comprisingpreparing an aqueous solution containinga protein having plasminogen activator activity, sugar, and tranexamicacid, freezing the aqueous solution, adjusting the temperature of thefrozen solution to a value below the glass temperature, and lyophilizingthe frozen solution in a vacuum.
 26. The method according to claim 25,wherein said temperature is adjusted to between -40° C. and -50° C. 27.The process according to claim 25, wherein prior to lyophilization theaqueous solution contains the protein having plasminogen activatoractivity at a concentration which is up to three times higher than theconcentration of a ready-to-use aqueous injection or infusion solution.28. A method for treating blood clots comprising administering to apatient in need of such treatment, an effective amount of a compositioncomprising an effective amount of a protein having plasminogen activatoractivity, sugar and tranexamic acid, wherein said tranexamic acid is theonly aminocarboxylic acid in said composition, said composition isdevoid of citrate and wherein said composition is in a vein-toleratedpharmaceutical administration form.
 29. A pharmaceutical compositioncomprising a protein having plasminogen activator activity, sugar and asingle aminocarboxylic acid, wherein said single aminocarboxylic acid istranexamic acid, and wherein said composition is devoid of citrate.